Int. J. Cancer: 52,286-289 (1992) 0 1992 Wiley-Liss, Inc.
4
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Publication of the InternationalUnion Against Cancer Publication de I’Union Internationale Contre le Cancer
EFFECTS OF SHORT-CHAIN FATTY ACIDS ON GROWTH AND DIFFERENTIATION OF THE HUMAN COLON-CANCER CELL LINE HT29 Laurence GAMET?,Daniele DAVIAUDI, Colette DENIS-POUXVIEL~, Christian REMESY?and Jean-Claude MURAT1,3 lINSERM, U 31 7, Britiment L3, CHU Ranpied, 31054 Toulouse;arid ?Laboratoiredes Maladies Mitabohques, INRA Theu; 63122 Ceyrat. France. Short-chain fatty acids (SCFAs), namely butyrate, acetate and propionate, originate from the bacterial fermentation of dietary fibers and are the predominant anions present in the large bowel. Our study was carried out to investigatethe effects of SCFAs on growth of the human adenocarcinoma cell line, HT29. The results show that, under our culture conditions, both propionate and butyrate inhibit growth of HT29 cells, whereas acetate has no significant effect. The antiproliferative effect of propionate or butyrate is associated with an inhibition of FCS-induced activation of ornithine decarboxylase (ODC), a key enzyme of polyamine metabolism. Inhibition of growth induced by either propionate or butyrate is not reversed by the addition of putrescine, which reveals that these SCFAs are not acting solely on the ODC/polyamine system. Our data show that propionate and butyrate, unlike acetate, induce an increase in alkaline phosphatase activity, which reflects a more differentiated phenotype than that of untreated control cells. Taken together, our results suggest that propionate, like butyrate, may play an important role in the physiology of the colon and could partially account for the protective effect of dietary fibers with respect to colon carcinogenesis. 8 1992 Wilq-Liss, Inc.
The proliferation of the epithelial cells from intestinal mucosa exhibits somewhat peculiar characteristics since it can be affected not only by gastrointestinal hormones and growth factors but also by various components present in the lumen and originating from the digestive process. Although the precise causes of colorectal cancer remain largely unknown, a number of epidemiological and experimental data indicate that the diet may be an important factor. As a result of these studies, some inteqtinal nutriments or metabolites, such as fat and secondary biliary acids, can be considered as tumor promoters. while others, such as fibers. appear to have a protective effect against colon cancer (Weisburger, 1991). As soon as they reach the large intestine, most of the carbohydrates of the fiber fraction are extensively broken down by the microflora. This fermentation process results in the production of gases and short-chain fatty acids (SCFAs), chiefly represented by acetic, propionic and butyric acids (Rtmesy and DemignC, 1976). The observation that dietary fibers may protect against large-bowel cancer is at least partially explained by the fact that they dilute the gut content and speed up the transit time, thus diminishing both the concentration and the time of exposure to carcinogens or promoters (Weisburger, 1991). However, it appears that the metabolic products of the fermentation of fibers may also be important in themselves. Among SCFAs, butyrate has given rise to a great deal of interest. Besides its role as an energetic substrate for the colonic mucosa (Roediger, 1982). butyrate was found to be an antiproliferative and differentiating agent in various cancer-cell lines in vitro (Kruh et al., 1991). O n the other hand, short-chain fatty acids have stimulatory effects on epithelial-cell proliferation in rat large intestine (Scheppach et al., 1992). Moreover, various fibers modulate large-bowel mucosal growth and cell proliferation (Jacobs and Lupton, 1984). We have studied the effects of SCFAs on growth and differentiation of the human colonic adenocarcinoma cell line HT29 and compared their effects to those of butyrate. The HT29 cell line was chosen for this study since it is a valuable
model for in vitro studies related to intestinal cell functions. Moreover, this line is able to undergo different patterns of intestinal differentiation depending on nutritional manipulations (Zweibaum el al., 1991). MATERIAL AND METHODS
Drugs and chemicals Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Eurobio (Paris, France) and FCS from IBF (Villeneuve-la-Garenne, France). IT-DL-ornithine was purchased from NEN/Dupont de Nemours (Boston, MA). Difluoromethylornithine (DFMO) was kindly provided by Merrell Dow (Strasbourg, France). All other chemicals were obtained from Sigma (St Louis, MO) or from Serva (Heidelberg, Germany) and were of the highest purity grade.
Cell culture The HT29 cell line was established in permanent culture from a human colonic adenocarcinoma by Dr. J. Fogh. In the present work, we used a HT29 cell subpopulation (HT29 Rev Glc-) selected by long-lasting adaptation to growth in a sugar-free medium (Zweibaum et al., 1985) and re-cultured in glucose-containing medium for more than 8 passages. This subpopulation, which is able to undergo spontaneous “enterocyte-like” post-confluence differentiation, will be referred to as HT29 Rev Glc-, or, more shortly, as HT29 cells. Routinely, stock cells were cultured at 3 7 T , under an air/COz (9:l) atmosphere, in DMEM containing 25 mM glucose, 60 pg/ml penicillin, 100 kg/ml streptomycin and 5% (v/v) heatinactivated FCS (standard medium). For the experiments, cells were seeded at low density (4 x lo4 cells per cm2) in 35-mm-diamcter plastic dishes in standard medium. One day after seeding, the cells were rinsed and placed in serum-free medium. Flow cytometric analysis of the D N A indicated that 90% of the cell population was synchronized in G o / G Iphase after a 24-hr period of FCS deprivation (Garnet et al., 1992). All the experiments were started by stimulating such growtharrested cells with 1 % FCS alone or in combination with either acetic, propionic or butyric acid. The effect of the various SCFAs on cell growth was estimated by cell counting and measurement of cellular protein content per dish. Cell counts Cell counts were performed 48 hr after the re-initiation of growth by FCS. The cells were detached by treatment of the cell layers with 0.25% trypsin-0.6 mM EDTA in PBS. A n aliquot of the cell suspension was diluted in Isoton I1 diluent and the cell number determined using a Coulter Counter (Les
)To whom correspondence and reprint requests should be sent. at INSERM. Toulouse. Fax: 61 33 17 21. Abbreviutions: SCFA, short-chain fatty acid; FCS, fetal calf serum; ODC, ornithine decarboxylase; DMEM, Dulbecco’s modified Eagle’s medium.
Received: February 14, 1992 and in revised form May 5 , 1992.
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Ulis, France). Determinations within each experimental group were done in quadruplicate.
Estimation of growth rate Growth rate was estimated by measuring the total protein content per dish. This parameter correlates linearly with the number of ct:lls. Growth-arrested cells (see “Cell culture”) were cultured in the presence of 1 % FCS and the indicated concentrations of each SCFA. Medium was changed daily and total protein content per dish was determined at the indicated time over a (2-day period of culture, using the method of Bradford (1976). Assay of omithirie decarboxylase (ODC) activity After removal of the culture medium, cell layers were rinsed with 2 ml of ice-cold 0.9% NaCI. The cells were harvested in 1 ml of ice-cold 10 mM Tris-HCI buffer (pH 7.5) containing 5 mM dithiothreitol and 0.1 mM EDTA, and sonicated twice for 5 sec on ice. The resulting homogenate was centrifuged at 38,000 g for 20 min and then used for the determination of ornithine decarboxylase (EC.4.1.1.17) activity, as described by Gamet et al. (1991). Briefly, 400 p1 of the supernatant were mixed with 100 pI (of an incubation medium containing 125 mM Tris-HCI (pH 7.5), 2.5 mM dithiothreitol, 0.2 mM pyridoxal-P, 0.16 mM L-ornithine and 0.25 pCi 14C-DL-ornithine as tracer. Incubation was run for 1 hr at 37”C, then stopped by addition of 200 pl 0.4 M HC104. The COz produced was trapped on a Whatrnan 3 MM filter previously impregnated with 1 M methylbenzethonium hydroxide in methanol. The filter was then placed in vials with 4 ml of scintillation liquid and counted for radioactivity with correction of luminescence and quenching. For each experimental point, the O D C activity was evaluated as the difference between total activity, measured in the absence of inhibitor, and non-specific activity, measured in the presence of 2 mM DFMO. Activity of O D C is expressed as pmol of putrescine (stochiometrically equivalent to COz) produced from ornithine/mg prot/hr, at 37°C. Assay of alkaline phosphatase Alkaline phosphatase activity was determined according to the method of Garen and Levinthal(l960). Cells were homogenized in 50 mM Tris buffer (pH 7.4) by several passages through a 26-gauge needle fitted to a 2-ml syringe. The conversion of p-nitrophenylphosphate to p-nitrophenol was studied at 37°C for 60 min by mixing 0.1 ml of cell homogenate with 0.5 ml of a 150-mM 2-amino-2-methyl 1-propanol buffer (pH 9.5) containing 1 mM MgS04 and 1.5 mM of p-nitrophenylphosphate. The reaction was stopped by the addition of 0.15 ml of NaOH 1 N. The amount of p-nitrophenol liberated was determined spectrophotometrically at 410 nM. The enzyme activity was expressed as mU per mg of cellular protein (one unit being one pmole of substrate hydrolyzed per min). RESULTS
Effects of acetate, propionate and butyrate on the growth of HT29 cells Quiescent HT29 cells, obtained by deprivation of FCS for 1 day, were re-initiated to growth with a suboptimal dose of FCS (1%) alone or in the presence of different concentrations of either acetic, propionic or butyric acid (Fig. 1). Estimation of cell number 48 hr after the treatment indicated that readdition of FCS triggered a 70% increase in cell population. This was not affected by the addition of acetate. By contrast, and as previously observed in other cell lines (Kruh et al., 1991), the addition of 2 mM butyrate resulted in a marked inhibition of the FCS-induced proliferation. Concentrations higher than 2 mM provoked significant cell mortality,
20 000
n
E .
2 E,
10000
-a C
0
+ FCS 1%
FIGURE1 - Effect of acetate, propionate and butyrate on cell proliferation. Quiescent HT29 cells were stimulated with 1% FCS alone (m) in the presence of either acetate (C2 N), propionate (C3 a),or butyrate (C4 EJ) at the indicated concentrations. Cell number was estimated after a 48-hr period of culture. Control cells were cultured in the absence of FCS during the experiment (0). Results are the means f SEM of 4 separate experiments. *Indicates a significant difference compared to FCS-stimulated cells (m) a t p < 0.01.
0
4 a Time (days)
12
FIGURE2 - Growth curves of HT29 cells in the presence of 1% FCS (0)plus either 10 mM acetate (A) or 5 mM propionate (0) or 2 mM butyrate ( 0 ) .Cell growth was estimated by measuring the total protein content of the dishes at the given times. Each curve is the mean of f SEM of different experiments. When they do not appear, error bars are smaller than the symbols.
suggesting that butyrate exerts cytotoxic effects on HT29 cells under these conditions. Our results also clearly show that propionate can exert an antiproliferative effect. Indeed, addition of 2 or 5 mM propionate resulted in about 60% inhibition of serum-induced cell growth. A stronger inhibition was observed at 10 mM, but treatment at such a concentration was also associated with some cellular mortality. With respect to these preliminary observations and to the physiological ratio of the SCFAs in the digestive tract (Roediger, 1982), further experiments were carried out using 2 mM butyrate, 5 mM propionate or 10 mM acetate. Cell proliferation was also estimated by measuring total cellular protein content per dish over a 12-day period of culture in the absence or presence of SCFAs. The results of this long-term experiment (Fig. 2) confirm unambiguously the antiproliferative effect of propionate. This effect is not due to any toxicity toward the cells, since glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities were unchanged in SCFA-treated cells compared to control cells
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GAMET E T A L .
during the course of the experiment (data not shown). Similar results were obtained with 2-mM butyrate-treated cells.
Propionate and butyrate inhibit the omithine decarboxylase activation induced by FCS In the HT29 cell line, as in all other models investigated so far (Tabor and Tabor, 1984), the production of polyamines appears to be a crucial process in the proliferative response of the cells. As previously shown (Garnet et al., 1991), addition of FCS to quiescent HT29 cells resulted in a rapid activation of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis. Maximal activation of this enzyme occurred 9 hr after addition of FCS. As shown in Figure 3, treatment of the cells with propionate for 24 hr prior to addition of 1% FCS resulted in 65% inhibition of serum-induced O D C activation. In butyrate-treated cells, the activity of O D C was as low as in growth-arrested cells (i.e., maintained in serum-free medium). By contrast, acetate did not significantly affect the enzyme activity. Thus, the effects of SCFAs on O D C activity are in agreement with their antiproliferative effects. Further experiments were designed to test whether inhibition of cell growth by butyrate and propionate was due to their negative effect on polyamine metabolism only. For that purpose, quiescent HT29 cells were stimulated with l % FCS and treated with each SCFA in the presence or absence of 10 p M putrescine, which directly provides polyamines in case of O D C inhibition (Tabor and Tabor, 1984). As shown in Figure 4, the addition of putrescine failed to reverse the inhibition of HT29 cell growth induced by propionate and butyrate. This result suggests that propionate, as well as butyrate, affects other biochemical processes involved in growth regulation.
alkaline phosphatase activity in several cell lines, including those derived from human colorectal cancer (Kruh et al., 1991). DISCUSSION
The protective effect of dietary fibers against colorectal cancer is now widely accepted. Nevertheless, the exact mechanism by which such dietary compounds prevent carcinogenesis of the large bowel is poorly understood. Short-chain fatty acids (SCFAs) are the predominant anions in human feces and originate from fiber fermentation which takes place in the colon. Despite their presence in the colon at rather high concentrations (about 100 mM), there is relatively little information regarding the effects of SCFAs on normal and malignant intestinal mucosa. Butyrate appears to be of special interest in colorectal cancer, since patients suffering from adenomatous polyps or colon cancer have a significantly higher incidence of low butyrate fermentation (Weaver et al., 1988). It has also been shown that butyric acid can inhibit the development of grafted tumors in mice (Okata et al., 1989). Given the difficulty of studying in vivo the antiproliferative influence of SCFAs on colon carcinogenesis, we have chosen to study their effect on an established cell line. So far, no study has reported growth-regulatory properties of the SCFAs, except in a human colon cancer cell line (LIM1215) in which the inhibitory effect of butyrate was confirmed (Whitehead et al., 1986). Propionate could indeed mimic the effects of butyrate on cell growth and differentiation of the HT29 colon adenocarcia
b T
60
Effect of SCFAs on HT29 cell differentiation In order to test whether the antiproliferative effect of propionate was correlated with an induction of cellular differentiation, the activity of alkaline phosphatase was measured. Figure 5 shows the evolution of alkaline phosphatase activity during a 12-day period of culture in cells treated with 1% FCS alone or in combination with either 10 mM acetate or 5 mM propionate or 2 mM butyrate. The level of alkaline phosphatase activity remained unchanged over the whole period of culture in control (1% FCS) cells (which are in the exponential growing phase) or in acetate-treated cells. Treatment with propionate led to a significant increase in this enzyme activity which, however, was less pronounced than that obtained with butyric acid which has been reported to increase the levels of
:E g
f
30
-c 8
0 TIME (days)
TIME (days)
FIGURE 4 -Effect of putrescine addition on (a) propionate and (b) butyrate-induced inhibition of cell growth. Quiescent cells were stimulated by 1% FCS and treated with either 5 mM propionate alone (B)or propionate in combination with 2 pM ) (a) or with 2 m M butyrate alone (69) or butyrate in combination with 2 KM putrescine (0)(b). Cell proliferation was estimated b measuring cell number at the indicated time. Black corresponds to cells exposed to 1% FCS but not histogram treated. Resu ts are the mean & SEM of 3 separate experiments.
(h,)
4000
2.
g
0
1.5
3000 2000 1000
0
none
C2
C3
C4
controls 0
FIGURE 3 - Effect of the different SCFAs on FCS-induced ODC activation. HT29 cells were incubated for 24 hr in serum-free medium prior to the addition of FCS with either 10 mM acetate (C2) or 5 mM propionate (C3) or 2 mM butyrate (C4). ODC activity was measured 9 hr after FCS addition. Enzyme activity is expressed as pmol of putrescine produced/mg prot./hr at 37°C. Black column corresponds to cells exposed to FCS but not treated with SCFA. Control cells (open column) were cultured in serumfree medium during the lag time of the experiment. Results are the mean 2 SEM of 4 separate experiments.
.
0
5 4
8
1 12
FIGURE 5 -Evolution pattern of alkaline phosphatase activity in HT29 cells cultured in the presence of FCS alone (0)or in the presence of either 10 mM acetate (A) or 5 mM propionate ( 0 )or 2 mM butyrate (0). Each curve is the mean f SEM of 3 different experiments. Specific enzyme activities are expressed as mU/mg of protein. *Indicates a significant difference p < 0.01 as compared to control (0). When they do not appear, error bars are smaller than the symbols.
PROPIONATE INHIBITS COLON-CANCER CELL PROLIFERATION
noma cell line. The concentrations of SCFAs used in our study are below those found in the lumen of the large bowel (Remesy and Demigne, 1976), suggesting that the observed effect may be of physiological relevance. Moreover, the effect of propionate on cell growth is correlated with inhibition of FCSinduced O D C activity. Similar but stronger effects are observed in butyrate-treated cells. Such a result is not surprising since, in various cell types, butyrate alters the activity of proteins and/or expression of several genes related to cell growth (cmyc. cfos,P53 and cdc2) (Charollais et al., 1990; Kruh et al., 1991). Addition of putrescine fails to reverse the inhibition of proliferation observed in propionate- or butyratetreated cells, indicating that their antiproliferative effects are not only due to an altered polyamine metabolism. Further work will be necessary to clarify the molecular mechanisms whereby propionate induces inhibition of HT29 cell growth. Indeed, it remains to be shown with which aspect of the mitogenic cascade propionate might interfere. The inhibition of growth, observed in propionate-treated cells, is associated with an induction of the alkaline phosphatase activity. In the small intestine, the enzyme is localized in the brush-border membrane of the epithelial cells and its activity is considerably higher in differentiated cells from the villus than in proliferative cells from the crypt (Dawson and Pyrsc-Davies, 1963). Malignant transformation of a variety of cells provokes a decrease in the activity of alkaline phosphatase (Sella and Sacks, 1974). The HT29cell line synthetizcs the intestinal form of alkaline phosphatase. The activity of the enzyme is low during the exponential phase of growth but can be enhanced by differentiating agents (Hertz et al., 1981). Therefore, the increase in activity, observed in propionatetreated HT29 cells, may reflect a more differentiated phenotype than that in the untreated cells and suggests that
289
propionate, like butyrate, could cause the HT29 cells to differentiate into absorptive cells. In all the experiments, acetate fails to affect cell growth and differentiation. However, in chick bone cells in culture, acetate was reported to inhibit cell growth and to stimulate alkaline phosphatase activity (Saitta et al., 1989). Moreover, in the LIM121.5 human colon carcinoma cells, acetate has little differentiating effect (Whitehead et al., 1986). The absence of an effect of acetate in our experiments may be due to a rapid metabolization of this SCFA by HT29 cells. It would be of interest to know whether the effect of propionate can be extrapolated to other human colon cancer cells and normal colon cells. In this respect, Scheppach et al. (1992) have shown that propionate and butyrate could promote the proliferation of normal intestinal cells. Luminal butyrate concentrations arc quite dependent on the availability of fermentable carbohydrates, whereas propionate concentrations are more constant (Titgemeyer et al., 1991). Our study suggests that both butyrate and propionate may play an important role in vivo to inhibit growth of neoplastic colonic cells. These acids could account for the protective effect of dietary fibers against colon carcinogenesis. It is possible to speculate that both butyrate and propionate could play a dual regulatory role on colonic function and structure, activating the normal mucosal growth rate and inhibiting neoplastic proliferation. ACKNOWLEDGEMENTS
This work was supported by the National Institute of Agronomic Research (INRA; grant A.I.P. 90/4725) and by the Association for the Research against Cancer (ARC, Villejuif; grant 6161 to J.C.M.).
REFERENCES BRADFORD, M.M., A rapid sensitive technique for the quantification of protein utilizing the principle of protein-dye binding. Anal. Biochern., 72,248-254 (1976). CHAROLLAIS, K.H., BIQUET,C. and MESTER.J., Butyrate blocks the accumulation of CDC:! mRNA in late GI phase but inhibits both the early and late G I progression in chemically transformed mouse fibroblast BP-A31.J. cell. Physiol., 145,46-52 (1990). DAWSON. I . and PYRSt-DAVIES, J., The distribution of certain enzyme systems in the normal human gastrointestinal tract. Gastroenterology, 44, 745-760 (1'363). GAMEI. L.. CAZENAVE, Y., TROCHERIS, V., DENIS-POUXVIEL, C. and MURAT, J.C., Involvement of ornithine decarboxylase in the control of proliferation of the human colon cancer cell line. Effect of vasoactive intestinal peptide on enzyme activity. Znr. J. Cancer, 47, 633-638 (1991). GAMET,L., MURAT.J.C.. REMAURY. A,, R ~ M E s YC.. , VALET.P., PARIS, H. and DENIS-POUXVIEL. C.. Vasoactive intestinal peptide and forskoline regulate rolifeiation of the HT29 human colon adenocarcinoma cell 1ine.J. c e l Physiol., 150,501-509 (1992). GAREN. A. and LEviNirtiAL, C.. A fine structure genetics and chemical study of the enzyme alkaline phosphatase of E. coli. Purification and characterization of alkaline phosphatase. Biochim. biophys. Acta, 38, 470-483 ( 1960). HERTZ,F., SC'HERMER, A., HALWER,M. and BOGART.L.H., Alkaline phosphatase in HT29.. a human colon cancer cell line: influence of sodium butyrate and hyperosmolality. Arch. Biocheni. Biophys., 210, 581-591 (1981). JAC'OW,L.R. and LWTON,J.R., Effects of dietary fiber on rat large bowel mucosa growth and cell proliferation. Amer. J. Pliy.~iol.,246, 378-385 (1984). KRUH,J., DEFEK, N. and TICHONICKY, L.. Molecular and cellular effects of sodium butyrate. Tenth Ross Conference on Medical Issus. Sliorr-chainj i q acids: tnetahohsnz and clinical irnj>orfance,pp. 45-50, (199 1). OKATA.M.. SINGHAL, A. and HAKOMORI, S., Antibody-mediated targetting of differentiation inducers to tumor cells: inhibition of colonic cancer cell growth in vitro and in v i i a Biophys. Biochem. Res. Coinin.. 158, 202-208 (1989).
R ~ M ~ sC. Y and , D E M I G NC., ~ , Partition and absorption of volatile fatty acids in the alimentary canal of the rat.Ann. Rech. Vkr., 7,39-55 (1976). ROEDIGER, W.E.. Utilization of nutrients by isolated epithelial cells of rat colon. Gasrroenterology. 83,423-429 (1982). SAITTA.J.C.N., LIPKIN, E.N. and HOWARD, G.A., Acetate inhibition of chick bone cell roliferation and bone growth in virro. J. Bone Miti. Res., 4, 379-386 5989). SCHEPPACH, W.. BARTRAM, P., RICHTER, A,, RICHTER. F., LIEPOLD, H., DUSEL,G., HOFSTETTER, G., RUTHLEIN, J. and KASPER,H., Effect of short chain fatty acids on the human colonic mucosa in vitro. J. Parenter. Enter. Nutr., 16,43-48 (1992). SELLA,B.A. and SACKS.L.. Alkaline Dhosuhatase activitv and the regulation of growth in transformed m~mmaiiancells. J. cek Physiol., 83,27-34 (1974). TABOR,W.E. and TABOR,H., Polyamines. Ann. Rev. Biochrm., 53, 749-790 (1984). TITGEMEYER, E.C.. BOURQUIN, L.D.. FAHEY,G.C. and GARLEB, K.A., Fermentability of various fiber sources by human fecal bacteria in v i t m Atner. J. clin. Nufr., 53, 1418-1424 (1991). WEAVER, G.A., KRAUSE,J.A., MILLER,T.L. and WOLLIN,M.J., Short chain fatty acid distributions of enema samples from a sigmoidoscopy population and association of high acetate and low butyrate ratios with adenomatous polyps and colon cancer. Gut, 29, 1535-1542 (1988). WEISBURGER. J.H., Causes, relevant mechanisms and prevention of the large bowel cancer. Semin. Oncol., 18,316-336 (1991). WHITEHEAD, R.H., YOUNG,G.P. and BHATHAL,P.S., Effects of short chain fatty acids on a human colon carcinoma cell line (LIM1215). Gut, 27,1457-1463 (1986). ZWEIBAUM, A., LABURTHE, M., GRASSET, E. and LOUVARD. D.. Use of cultured cell lines in studies of intestinal cell differentiation and function. h i : M. Field and R.A. Frizzel (eds.), Handbook ofphysiology, Section 6: the gastrointestinal system, Vol. Iv: htminal absorprion and srcretion, pp. 22.%235, American PhysiologicalSociety, Bethesda (1991). ZWEIBAUM. A,, PINTO,M., CHEVALLIER, G., DUSSAULX, E., TRIADOU, N.. LACROIX, B., HAFFEN,K., BRUN,J.L. and ROUSSET,M., Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT29 selected for growth in sugar-free medium and its inhibition by glucose. J. cell. fliysiol., 122,21-29 (1985).
4
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Publication of the InternationalUnion Against Cancer Publication de I’Union Internationale Contre le Cancer
EFFECTS OF SHORT-CHAIN FATTY ACIDS ON GROWTH AND DIFFERENTIATION OF THE HUMAN COLON-CANCER CELL LINE HT29 Laurence GAMET?,Daniele DAVIAUDI, Colette DENIS-POUXVIEL~, Christian REMESY?and Jean-Claude MURAT1,3 lINSERM, U 31 7, Britiment L3, CHU Ranpied, 31054 Toulouse;arid ?Laboratoiredes Maladies Mitabohques, INRA Theu; 63122 Ceyrat. France. Short-chain fatty acids (SCFAs), namely butyrate, acetate and propionate, originate from the bacterial fermentation of dietary fibers and are the predominant anions present in the large bowel. Our study was carried out to investigatethe effects of SCFAs on growth of the human adenocarcinoma cell line, HT29. The results show that, under our culture conditions, both propionate and butyrate inhibit growth of HT29 cells, whereas acetate has no significant effect. The antiproliferative effect of propionate or butyrate is associated with an inhibition of FCS-induced activation of ornithine decarboxylase (ODC), a key enzyme of polyamine metabolism. Inhibition of growth induced by either propionate or butyrate is not reversed by the addition of putrescine, which reveals that these SCFAs are not acting solely on the ODC/polyamine system. Our data show that propionate and butyrate, unlike acetate, induce an increase in alkaline phosphatase activity, which reflects a more differentiated phenotype than that of untreated control cells. Taken together, our results suggest that propionate, like butyrate, may play an important role in the physiology of the colon and could partially account for the protective effect of dietary fibers with respect to colon carcinogenesis. 8 1992 Wilq-Liss, Inc.
The proliferation of the epithelial cells from intestinal mucosa exhibits somewhat peculiar characteristics since it can be affected not only by gastrointestinal hormones and growth factors but also by various components present in the lumen and originating from the digestive process. Although the precise causes of colorectal cancer remain largely unknown, a number of epidemiological and experimental data indicate that the diet may be an important factor. As a result of these studies, some inteqtinal nutriments or metabolites, such as fat and secondary biliary acids, can be considered as tumor promoters. while others, such as fibers. appear to have a protective effect against colon cancer (Weisburger, 1991). As soon as they reach the large intestine, most of the carbohydrates of the fiber fraction are extensively broken down by the microflora. This fermentation process results in the production of gases and short-chain fatty acids (SCFAs), chiefly represented by acetic, propionic and butyric acids (Rtmesy and DemignC, 1976). The observation that dietary fibers may protect against large-bowel cancer is at least partially explained by the fact that they dilute the gut content and speed up the transit time, thus diminishing both the concentration and the time of exposure to carcinogens or promoters (Weisburger, 1991). However, it appears that the metabolic products of the fermentation of fibers may also be important in themselves. Among SCFAs, butyrate has given rise to a great deal of interest. Besides its role as an energetic substrate for the colonic mucosa (Roediger, 1982). butyrate was found to be an antiproliferative and differentiating agent in various cancer-cell lines in vitro (Kruh et al., 1991). O n the other hand, short-chain fatty acids have stimulatory effects on epithelial-cell proliferation in rat large intestine (Scheppach et al., 1992). Moreover, various fibers modulate large-bowel mucosal growth and cell proliferation (Jacobs and Lupton, 1984). We have studied the effects of SCFAs on growth and differentiation of the human colonic adenocarcinoma cell line HT29 and compared their effects to those of butyrate. The HT29 cell line was chosen for this study since it is a valuable
model for in vitro studies related to intestinal cell functions. Moreover, this line is able to undergo different patterns of intestinal differentiation depending on nutritional manipulations (Zweibaum el al., 1991). MATERIAL AND METHODS
Drugs and chemicals Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Eurobio (Paris, France) and FCS from IBF (Villeneuve-la-Garenne, France). IT-DL-ornithine was purchased from NEN/Dupont de Nemours (Boston, MA). Difluoromethylornithine (DFMO) was kindly provided by Merrell Dow (Strasbourg, France). All other chemicals were obtained from Sigma (St Louis, MO) or from Serva (Heidelberg, Germany) and were of the highest purity grade.
Cell culture The HT29 cell line was established in permanent culture from a human colonic adenocarcinoma by Dr. J. Fogh. In the present work, we used a HT29 cell subpopulation (HT29 Rev Glc-) selected by long-lasting adaptation to growth in a sugar-free medium (Zweibaum et al., 1985) and re-cultured in glucose-containing medium for more than 8 passages. This subpopulation, which is able to undergo spontaneous “enterocyte-like” post-confluence differentiation, will be referred to as HT29 Rev Glc-, or, more shortly, as HT29 cells. Routinely, stock cells were cultured at 3 7 T , under an air/COz (9:l) atmosphere, in DMEM containing 25 mM glucose, 60 pg/ml penicillin, 100 kg/ml streptomycin and 5% (v/v) heatinactivated FCS (standard medium). For the experiments, cells were seeded at low density (4 x lo4 cells per cm2) in 35-mm-diamcter plastic dishes in standard medium. One day after seeding, the cells were rinsed and placed in serum-free medium. Flow cytometric analysis of the D N A indicated that 90% of the cell population was synchronized in G o / G Iphase after a 24-hr period of FCS deprivation (Garnet et al., 1992). All the experiments were started by stimulating such growtharrested cells with 1 % FCS alone or in combination with either acetic, propionic or butyric acid. The effect of the various SCFAs on cell growth was estimated by cell counting and measurement of cellular protein content per dish. Cell counts Cell counts were performed 48 hr after the re-initiation of growth by FCS. The cells were detached by treatment of the cell layers with 0.25% trypsin-0.6 mM EDTA in PBS. A n aliquot of the cell suspension was diluted in Isoton I1 diluent and the cell number determined using a Coulter Counter (Les
)To whom correspondence and reprint requests should be sent. at INSERM. Toulouse. Fax: 61 33 17 21. Abbreviutions: SCFA, short-chain fatty acid; FCS, fetal calf serum; ODC, ornithine decarboxylase; DMEM, Dulbecco’s modified Eagle’s medium.
Received: February 14, 1992 and in revised form May 5 , 1992.
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PROPIONATE INHIBITS COLON-CANCER CELL PROLIFERATION
Ulis, France). Determinations within each experimental group were done in quadruplicate.
Estimation of growth rate Growth rate was estimated by measuring the total protein content per dish. This parameter correlates linearly with the number of ct:lls. Growth-arrested cells (see “Cell culture”) were cultured in the presence of 1 % FCS and the indicated concentrations of each SCFA. Medium was changed daily and total protein content per dish was determined at the indicated time over a (2-day period of culture, using the method of Bradford (1976). Assay of omithirie decarboxylase (ODC) activity After removal of the culture medium, cell layers were rinsed with 2 ml of ice-cold 0.9% NaCI. The cells were harvested in 1 ml of ice-cold 10 mM Tris-HCI buffer (pH 7.5) containing 5 mM dithiothreitol and 0.1 mM EDTA, and sonicated twice for 5 sec on ice. The resulting homogenate was centrifuged at 38,000 g for 20 min and then used for the determination of ornithine decarboxylase (EC.4.1.1.17) activity, as described by Gamet et al. (1991). Briefly, 400 p1 of the supernatant were mixed with 100 pI (of an incubation medium containing 125 mM Tris-HCI (pH 7.5), 2.5 mM dithiothreitol, 0.2 mM pyridoxal-P, 0.16 mM L-ornithine and 0.25 pCi 14C-DL-ornithine as tracer. Incubation was run for 1 hr at 37”C, then stopped by addition of 200 pl 0.4 M HC104. The COz produced was trapped on a Whatrnan 3 MM filter previously impregnated with 1 M methylbenzethonium hydroxide in methanol. The filter was then placed in vials with 4 ml of scintillation liquid and counted for radioactivity with correction of luminescence and quenching. For each experimental point, the O D C activity was evaluated as the difference between total activity, measured in the absence of inhibitor, and non-specific activity, measured in the presence of 2 mM DFMO. Activity of O D C is expressed as pmol of putrescine (stochiometrically equivalent to COz) produced from ornithine/mg prot/hr, at 37°C. Assay of alkaline phosphatase Alkaline phosphatase activity was determined according to the method of Garen and Levinthal(l960). Cells were homogenized in 50 mM Tris buffer (pH 7.4) by several passages through a 26-gauge needle fitted to a 2-ml syringe. The conversion of p-nitrophenylphosphate to p-nitrophenol was studied at 37°C for 60 min by mixing 0.1 ml of cell homogenate with 0.5 ml of a 150-mM 2-amino-2-methyl 1-propanol buffer (pH 9.5) containing 1 mM MgS04 and 1.5 mM of p-nitrophenylphosphate. The reaction was stopped by the addition of 0.15 ml of NaOH 1 N. The amount of p-nitrophenol liberated was determined spectrophotometrically at 410 nM. The enzyme activity was expressed as mU per mg of cellular protein (one unit being one pmole of substrate hydrolyzed per min). RESULTS
Effects of acetate, propionate and butyrate on the growth of HT29 cells Quiescent HT29 cells, obtained by deprivation of FCS for 1 day, were re-initiated to growth with a suboptimal dose of FCS (1%) alone or in the presence of different concentrations of either acetic, propionic or butyric acid (Fig. 1). Estimation of cell number 48 hr after the treatment indicated that readdition of FCS triggered a 70% increase in cell population. This was not affected by the addition of acetate. By contrast, and as previously observed in other cell lines (Kruh et al., 1991), the addition of 2 mM butyrate resulted in a marked inhibition of the FCS-induced proliferation. Concentrations higher than 2 mM provoked significant cell mortality,
20 000
n
E .
2 E,
10000
-a C
0
+ FCS 1%
FIGURE1 - Effect of acetate, propionate and butyrate on cell proliferation. Quiescent HT29 cells were stimulated with 1% FCS alone (m) in the presence of either acetate (C2 N), propionate (C3 a),or butyrate (C4 EJ) at the indicated concentrations. Cell number was estimated after a 48-hr period of culture. Control cells were cultured in the absence of FCS during the experiment (0). Results are the means f SEM of 4 separate experiments. *Indicates a significant difference compared to FCS-stimulated cells (m) a t p < 0.01.
0
4 a Time (days)
12
FIGURE2 - Growth curves of HT29 cells in the presence of 1% FCS (0)plus either 10 mM acetate (A) or 5 mM propionate (0) or 2 mM butyrate ( 0 ) .Cell growth was estimated by measuring the total protein content of the dishes at the given times. Each curve is the mean of f SEM of different experiments. When they do not appear, error bars are smaller than the symbols.
suggesting that butyrate exerts cytotoxic effects on HT29 cells under these conditions. Our results also clearly show that propionate can exert an antiproliferative effect. Indeed, addition of 2 or 5 mM propionate resulted in about 60% inhibition of serum-induced cell growth. A stronger inhibition was observed at 10 mM, but treatment at such a concentration was also associated with some cellular mortality. With respect to these preliminary observations and to the physiological ratio of the SCFAs in the digestive tract (Roediger, 1982), further experiments were carried out using 2 mM butyrate, 5 mM propionate or 10 mM acetate. Cell proliferation was also estimated by measuring total cellular protein content per dish over a 12-day period of culture in the absence or presence of SCFAs. The results of this long-term experiment (Fig. 2) confirm unambiguously the antiproliferative effect of propionate. This effect is not due to any toxicity toward the cells, since glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities were unchanged in SCFA-treated cells compared to control cells
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GAMET E T A L .
during the course of the experiment (data not shown). Similar results were obtained with 2-mM butyrate-treated cells.
Propionate and butyrate inhibit the omithine decarboxylase activation induced by FCS In the HT29 cell line, as in all other models investigated so far (Tabor and Tabor, 1984), the production of polyamines appears to be a crucial process in the proliferative response of the cells. As previously shown (Garnet et al., 1991), addition of FCS to quiescent HT29 cells resulted in a rapid activation of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis. Maximal activation of this enzyme occurred 9 hr after addition of FCS. As shown in Figure 3, treatment of the cells with propionate for 24 hr prior to addition of 1% FCS resulted in 65% inhibition of serum-induced O D C activation. In butyrate-treated cells, the activity of O D C was as low as in growth-arrested cells (i.e., maintained in serum-free medium). By contrast, acetate did not significantly affect the enzyme activity. Thus, the effects of SCFAs on O D C activity are in agreement with their antiproliferative effects. Further experiments were designed to test whether inhibition of cell growth by butyrate and propionate was due to their negative effect on polyamine metabolism only. For that purpose, quiescent HT29 cells were stimulated with l % FCS and treated with each SCFA in the presence or absence of 10 p M putrescine, which directly provides polyamines in case of O D C inhibition (Tabor and Tabor, 1984). As shown in Figure 4, the addition of putrescine failed to reverse the inhibition of HT29 cell growth induced by propionate and butyrate. This result suggests that propionate, as well as butyrate, affects other biochemical processes involved in growth regulation.
alkaline phosphatase activity in several cell lines, including those derived from human colorectal cancer (Kruh et al., 1991). DISCUSSION
The protective effect of dietary fibers against colorectal cancer is now widely accepted. Nevertheless, the exact mechanism by which such dietary compounds prevent carcinogenesis of the large bowel is poorly understood. Short-chain fatty acids (SCFAs) are the predominant anions in human feces and originate from fiber fermentation which takes place in the colon. Despite their presence in the colon at rather high concentrations (about 100 mM), there is relatively little information regarding the effects of SCFAs on normal and malignant intestinal mucosa. Butyrate appears to be of special interest in colorectal cancer, since patients suffering from adenomatous polyps or colon cancer have a significantly higher incidence of low butyrate fermentation (Weaver et al., 1988). It has also been shown that butyric acid can inhibit the development of grafted tumors in mice (Okata et al., 1989). Given the difficulty of studying in vivo the antiproliferative influence of SCFAs on colon carcinogenesis, we have chosen to study their effect on an established cell line. So far, no study has reported growth-regulatory properties of the SCFAs, except in a human colon cancer cell line (LIM1215) in which the inhibitory effect of butyrate was confirmed (Whitehead et al., 1986). Propionate could indeed mimic the effects of butyrate on cell growth and differentiation of the HT29 colon adenocarcia
b T
60
Effect of SCFAs on HT29 cell differentiation In order to test whether the antiproliferative effect of propionate was correlated with an induction of cellular differentiation, the activity of alkaline phosphatase was measured. Figure 5 shows the evolution of alkaline phosphatase activity during a 12-day period of culture in cells treated with 1% FCS alone or in combination with either 10 mM acetate or 5 mM propionate or 2 mM butyrate. The level of alkaline phosphatase activity remained unchanged over the whole period of culture in control (1% FCS) cells (which are in the exponential growing phase) or in acetate-treated cells. Treatment with propionate led to a significant increase in this enzyme activity which, however, was less pronounced than that obtained with butyric acid which has been reported to increase the levels of
:E g
f
30
-c 8
0 TIME (days)
TIME (days)
FIGURE 4 -Effect of putrescine addition on (a) propionate and (b) butyrate-induced inhibition of cell growth. Quiescent cells were stimulated by 1% FCS and treated with either 5 mM propionate alone (B)or propionate in combination with 2 pM ) (a) or with 2 m M butyrate alone (69) or butyrate in combination with 2 KM putrescine (0)(b). Cell proliferation was estimated b measuring cell number at the indicated time. Black corresponds to cells exposed to 1% FCS but not histogram treated. Resu ts are the mean & SEM of 3 separate experiments.
(h,)
4000
2.
g
0
1.5
3000 2000 1000
0
none
C2
C3
C4
controls 0
FIGURE 3 - Effect of the different SCFAs on FCS-induced ODC activation. HT29 cells were incubated for 24 hr in serum-free medium prior to the addition of FCS with either 10 mM acetate (C2) or 5 mM propionate (C3) or 2 mM butyrate (C4). ODC activity was measured 9 hr after FCS addition. Enzyme activity is expressed as pmol of putrescine produced/mg prot./hr at 37°C. Black column corresponds to cells exposed to FCS but not treated with SCFA. Control cells (open column) were cultured in serumfree medium during the lag time of the experiment. Results are the mean 2 SEM of 4 separate experiments.
.
0
5 4
8
1 12
FIGURE 5 -Evolution pattern of alkaline phosphatase activity in HT29 cells cultured in the presence of FCS alone (0)or in the presence of either 10 mM acetate (A) or 5 mM propionate ( 0 )or 2 mM butyrate (0). Each curve is the mean f SEM of 3 different experiments. Specific enzyme activities are expressed as mU/mg of protein. *Indicates a significant difference p < 0.01 as compared to control (0). When they do not appear, error bars are smaller than the symbols.
PROPIONATE INHIBITS COLON-CANCER CELL PROLIFERATION
noma cell line. The concentrations of SCFAs used in our study are below those found in the lumen of the large bowel (Remesy and Demigne, 1976), suggesting that the observed effect may be of physiological relevance. Moreover, the effect of propionate on cell growth is correlated with inhibition of FCSinduced O D C activity. Similar but stronger effects are observed in butyrate-treated cells. Such a result is not surprising since, in various cell types, butyrate alters the activity of proteins and/or expression of several genes related to cell growth (cmyc. cfos,P53 and cdc2) (Charollais et al., 1990; Kruh et al., 1991). Addition of putrescine fails to reverse the inhibition of proliferation observed in propionate- or butyratetreated cells, indicating that their antiproliferative effects are not only due to an altered polyamine metabolism. Further work will be necessary to clarify the molecular mechanisms whereby propionate induces inhibition of HT29 cell growth. Indeed, it remains to be shown with which aspect of the mitogenic cascade propionate might interfere. The inhibition of growth, observed in propionate-treated cells, is associated with an induction of the alkaline phosphatase activity. In the small intestine, the enzyme is localized in the brush-border membrane of the epithelial cells and its activity is considerably higher in differentiated cells from the villus than in proliferative cells from the crypt (Dawson and Pyrsc-Davies, 1963). Malignant transformation of a variety of cells provokes a decrease in the activity of alkaline phosphatase (Sella and Sacks, 1974). The HT29cell line synthetizcs the intestinal form of alkaline phosphatase. The activity of the enzyme is low during the exponential phase of growth but can be enhanced by differentiating agents (Hertz et al., 1981). Therefore, the increase in activity, observed in propionatetreated HT29 cells, may reflect a more differentiated phenotype than that in the untreated cells and suggests that
289
propionate, like butyrate, could cause the HT29 cells to differentiate into absorptive cells. In all the experiments, acetate fails to affect cell growth and differentiation. However, in chick bone cells in culture, acetate was reported to inhibit cell growth and to stimulate alkaline phosphatase activity (Saitta et al., 1989). Moreover, in the LIM121.5 human colon carcinoma cells, acetate has little differentiating effect (Whitehead et al., 1986). The absence of an effect of acetate in our experiments may be due to a rapid metabolization of this SCFA by HT29 cells. It would be of interest to know whether the effect of propionate can be extrapolated to other human colon cancer cells and normal colon cells. In this respect, Scheppach et al. (1992) have shown that propionate and butyrate could promote the proliferation of normal intestinal cells. Luminal butyrate concentrations arc quite dependent on the availability of fermentable carbohydrates, whereas propionate concentrations are more constant (Titgemeyer et al., 1991). Our study suggests that both butyrate and propionate may play an important role in vivo to inhibit growth of neoplastic colonic cells. These acids could account for the protective effect of dietary fibers against colon carcinogenesis. It is possible to speculate that both butyrate and propionate could play a dual regulatory role on colonic function and structure, activating the normal mucosal growth rate and inhibiting neoplastic proliferation. ACKNOWLEDGEMENTS
This work was supported by the National Institute of Agronomic Research (INRA; grant A.I.P. 90/4725) and by the Association for the Research against Cancer (ARC, Villejuif; grant 6161 to J.C.M.).
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